| Glioblastoma is an aggressive tumor characterized by extensive brain invasion. This infiltrative nature makes curative surgical resection hardly possible and contributes to the short median survival of glioblastoma patients. Meanwhile,the importance of angiogenesis in glioblastoma growth was recognized many decades ago. Angiogenesis and glioblastoma invasion share common features, at least in their initial stages, as they both require controlled degradation of extracellular matrix(ECM) components in order to allow tumor cell and vascular endothelial cell migration, as well as facilitate neovascularization and tumor infiltration. MMPs play a fundamental role in these processes. Since type IV collagen is the major component of the basement membrane, MMP-9 specifically targets type IV collagen and appears to play a crucial role in glioblastoma invasion and angiogenesis.In this study, firstly, in vitro we observe MMP-9 expression in a human glioblastoma cell line TJ905 transfected an antisense vector capable of expressing an antisense transcript complementary to a 528-bp segment at the 5' end of human MMP-9 cDNA. Followed, in vivo we investigate the effect of antiangiogenesis by the inhibition of MMP-9 expression and/or subcuticular injection of endostatin.Protocol of the present studyIn the first part of this study, a 528-bp cDNA fragment of MMP-9 was amplified by RT-PCR with synthetic primers and subcloned into the pcDNA3.0 vector between the Hind IIIand Xho I polycloning sites in the sense and antisense orientations. Recombination plasmids were analysed by restriction endonuclease and sequence analysis. Then TJ905 cells were transfected with sense construct, antisense construct, and pcDNA3.0 by using Lipofectamine 2000. Stable transfectants were selected by growing cells in G418; Expression of MMP-9 RNA and protein in the positive clones that survived were detected by RT-PCR and Western Blot. MMP-9 and Ki-67 expression of the positive cloneswere detected by immunohistochemistry.In the second part, in vivo study was carried out. A human glioblastoma cell line U251 cells were implanted subcuticularly into four to six-week old BALB/c nude mice. By heterotransplantation, mice with subcutaneous glioma were randomly divided into eight groups: treated with pcDNA3.0 empty vector, antisense MMP-9 construct, injected i.p. endostatin, transfected with antisense construct combined with injected i.p. endostatin, as well as untreatment mice as control. Each group consisted of five mice. Tumor size was measured with a digital vernier caliper three times weekly; at the end of four weeks and eight weeks respectively after treatment, mice were sacrificed and tumor weight was measured. Histopathological changes of tumor and expression of MMP-9 and Ki-67 in glioma cells were detected, as well as microvessel density of tumor was counted.ResultsRestriction endonuclease analysis and sequence analysis of recombination plasmids verified its 528-bp internal sequence 100% homology with the published sequence of MMP-9 cDNA.In vitro studyTJ905 glioma cells transfected with pcDNA3.0 vector, sense construct and antisense construct form stable transfectants after G418 selection. MMP-9 mRNA and protein levels in stable transfectants were identified by RT-PCR and Western Blot. Compared with control, vector-transfected clones and sense MMP-9 construct-transfected clones, antisense MMP-9 construct-transfected TJ905 cells reduced expression of MMP-9 mRNA and protein(P<0.001). On immunohistochemical examination, in antisense MMP-9 construct-transfected TJ905 cells, the proliferation activity was inhibited. No significant difference in the proliferation activity was observed among vector-transfected clones, sense MMP-9 construct-transfected clones and control.In vivo studyExcept two mice of U251 empty vector group were dead in six to seven weeks, other mice were alive until sacrificed. The growth velocity of tumor was significantly faster in control and empty vector groups than in other therapy...
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